Disarming of type I-F CRISPR-Cas surveillance complex by anti-CRISPR proteins AcrIF6 and AcrIF9

CRISPR-Cas systems are prokaryotic adaptive immune systems that protect against phages and other invading nucleic acids. The evolutionary arms race between prokaryotes and phages gave rise to phage anti-CRISPR (Acr) proteins that act as a counter defence against CRISPR-Cas systems by inhibiting the effector complex. Here, we used a combination of bulk biochemical experiments, X-ray crystallography and single-molecule techniques to explore the inhibitory activity of AcrIF6 and AcrIF9 proteins against the type I-F CRISPR-Cas system from Aggregatibacter actinomycetemcomitans (Aa). We showed that AcrIF6 and AcrIF9 proteins hinder Aa-Cascade complex binding to target DNA. We solved a crystal structure of Aa1-AcrIF9 protein, which differ from other known AcrIF9 proteins by an additional structurally important loop presumably involved in the interaction with Cascade. We revealed that AcrIF9 association with Aa-Cascade promotes its binding to off-target DNA sites, which facilitates inhibition of CRISPR-Cas protection.

3M). To homogeneously cover the PDMS surface with a film of the protein ink a 60 µl drop of 0.17 mg/ml traptavidin (tAv) protein solution (in buffer A) was placed on the stamp surface, mixed with the tip of pipette, and kept for 10 min. After incubation the protein ink was removed from the stamp by sucking it out with the pipette tip. Then, the stamp on glass slide was held with tweezers and washed with 5 ml of buffer A using a 1 ml pipette, ~50 ml ultrapure water using a wash bottle, and dried under N2 gas stream. For the printing procedure we employed a semi-automated printing machine [2]. First, the cleaned Si master was placed on the silicon rubber on the bottom of the printing machine and the pressure of 0.6 ml for both printing steps on Si master and silanized/PEGylated glass coverslip. The patterned glass coverslip was assembled into the flowcell, which was prepared as described earlier [1].
To immobilize the bt-λ DNA-dig the channel of the flowcell was filled with buffer B. To enhance surface passivation against non-specific protein adsorption, we injected 5% (v/v) Tween-20 solution in buffer B into the channel of the flowcell, incubated for 10 min and washed out with 600 µl of buffer A.
Next, the DNA (~40 pM 150 µl, in buffer B) was injected and incubated for at least 15 min. The excess of unbound DNA was washed out with ~300 µl of buffer A. Then DNA was labelled with the DNA intercalating green fluorescent dye -SYTOX green (SG, ThermoFisher Scientific, USA) at a concentration of ~0.4 nM (in buffer C). The SG dye was present during the entire time of the experiment. For the second DNA end tethering, the close-loop circulation was employed and 5 µl of biotin-anti-dig (bt-anti-dig) antibody was added (this resulted in ~0.05 mg/ml concentration) and incubated for at least 10 min at low speed (~0.1 ml/min). After 10 min, the speed of the buffer flow was increased to ~1 ml/min and kept constant for 20 min. Then, to remove the excess of unbound bt-anti-dig the flowcell was washed with 500 µl of buffer A in the open-loop circulation. Finally, 100 µl of imaging buffer was injected into the flowcell in order to reveal bound DNA.
Expression and purification of traptavidin and monovalent streptavidin. We followed previously published protocols for traptavidin (tAv) and monovalent streptavidin (mSav) preparation [3]. E. coli BL21 (DE3) strain cells were transformed with pET21a tAv or pET21a streptavidin (sAv) (Alive or Dead) plasmids, plated onto Luria-Broth (LB)-Carbenicillin agar plates and incubated at 37 °C overnight. An overnight culture, which was prepared by inoculating a colony from the agar plate in LB-Ampicillin (Amp) medium and shaking at 220 r.p.m. and 37 °C overnight, was then diluted 100-fold into LB-Amp, grown at 37 °C until OD600 0.9, induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside and shaken further at 37 °C for 4 h. After spinning the bacteria at 5000g and 4 °C for 10 min, the cell pellet was resuspended in a lysis buffer (50 mM Tris-HCl (pH 7.8), 300 mM NaCl, 5 mM EDTA, 0.8 mg/ml lysozyme, 1% (v/v) Triton X-100) and then put on a rocker at 80 r.p.m. and RT for 20 min. Pulsed sonication of the cell pellet on ice at 30% amplitude was performed afterwards for 10 min.
Centrifugation at 27000 g and 4 °C for 15 min was followed by three-times washing of the inclusion body pellet in a wash buffer (50 mM Tris-HCl (pH 7.8), 100 mM NaCl, 0.5% (v/v) Triton X-100). Isolated inclusion bodies were dissolved in 6 M guanidinium hydrochloride (GuHCl) (pH 1.5) and then spun at 17700 g and 4 °C for 20 min. Solubilized Alive and Dead sAv subunits were accordingly mixed to obtain a 5-fold molar excess of Dead sAv subunit in respect to Alive subunit. The resultant mixture of two different sAv subunits and GuHCl-dissolved tAv monomers were separately refolded by diluting them rapidly into PBS at 4 °C and stirring overnight at the same temperature. Protein precipitation using solid ammonium sulfate was then carried out in order to precipitate sAv and tAv from their refolds. The obtained precipitates were resuspended in a minimum volume of PBS at room temperature, centrifuged at 14000 g and 4 °C for 5 min and the excess of ammonium sulfate was removed by running the supernatant through a NAP-25 column. GE AKTA Prime Plus liquid chromatography system was used to purify tAv and monovalent sAv (mSav). Mixture of distinct sAv tetramers was loaded on the equilibrated HiTrap Chelating HP 5 ml column and mSav was then eluted by applying gradient elution.
An elution buffer (50 mM Tris-HCl (pH 7.8), 300 mM NaCl, 0.5 M imidazole) along with a start buffer (50 mM Tris-HCl (pH 7.8), 300 mM NaCl) was used during this procedure. Same steps were followed in the case of affinity-based tAv purification. After pooling the fractions containing tAv or mSav, the resultant protein solution was dialyzed one time in PBS at 4 °C overnight. Purified proteins were concentrated by using a 9 kDa MWCO centrifugal concentrator and spinning at 4800g and 4 °C for 20-30 min. The obtained final yields of tAv and mSav were ~ 2 mg/l and ~ 3 mg/l of initial culture, respectively.
We labelled the mSav using NHS-ATTO647N ester. We mixed 10 µM of mSav with 20 µM of NHS-ATTO647N (dissolved in DMSO (dimethyl sulfoxide)) in PBS and incubated at RT for 1 hour. After incubation, we quenched the excess dye using glycine. Then, we purified the protein using NAP-5 column (GE Healthcare). The purified protein had ~1 µM concentration.
The volume analysis of DNA-protein complexes. Atomic force microscopy produce threedimensional topological images. The heights and diameters of a number of particles were determined and used to calculate molecular volumes (Eq. 1). Particle diameter was measured at half the maximal height.
Where h is the height of the protein particle, d is its diameter. The theoretical volume is determined by Eq. 2: Here, MW is the molecular weight, NA is Avogadro's number, and V1 and V2 are the partial specific volumes of the individual protein 0.74 cm 3 g -1 and 1 cm 3 g -1 water, respectively. d is the extent of protein hydration (0.4 mol H2O/mol protein) [4].
Mean values for particle molecular volume are given in Supplementary Table S4.